Isolated hOAT polypeptide

ABSTRACT

This invention is concerned with human organic anion transporter (“hOAT”). Isolated nucleic acid encoding hOAT is provided, along with ioslated hOAT polypeptide. hOAT nucleic acid and/or hOAT polypeptide are employed in transgenic animals, recombinant cells, replicable vectors and analytical procedures for identifying hOAT agonists or antagonists, assays for identifying hOAT alleles and/or isotypes, screening tests for nephrotoxic or neurologically active compounds, and determination of drug-drug interactions within the kidney or brain.

This application is a Divisional of Ser. No. 09/330,245 filed Jun. 10,1999, now U.S. Pat. No. 6,432,631, which claims benefit of 60/088,864filed Jun. 11, 1998, and claims benefit of 60/132,267 filed May 03,1999.

BACKGROUND OF THE INVENTION

This invention relates to the processing of anions by the human kidney.In particular, it relates to nucleic acids and polypeptides transcribedand expressed in the kidneys that are responsible for the removal oftoxic anions from the circulation.

Sweet et al. have described a rat gene (ROAT1) encoding an organic aniontransport protein expressed in the rat kidney (Journal of BiologicalChemistry 272:30088–30095 [1997]). Sekine et al. also appear to havedisclosed essentially the same gene, denominated OAT1 (Journal ofBiological Chemistry 272:18526–18529 [1997]). Its apparent murinecounterpart has been described in Lopez-Nieto et al. Journal ofBiological Chem. 272:6471–6478 (1997).

It is an object of this invention to isolate nucleic acid encoding ahuman organic anion transporter (hOAT).

It is another object of this invention to recombinantly express nucleicacid encoding hOAT.

Another object is to obtain hOAT expressed in elevated yields inrecombinant cell culture.

An object is to prepare isolated polypeptide encoding hOAT.

An additional object of this invention is to provide novel hOATpolypeptides.

A further object of this invention is an assay for evaluation ofpotential drug-drug interactions due to interference of one drug orgroup of drugs with hOAT-mediated active kidney excretion of anotherdrug.

A further object of this invention is an assay system for screeningcandidate compounds that agonize or antagonize the expression of hOATand/or the biological activity of hOAT polypeptide, especially its aniontransport activity.

Another object is to provide an hOAT screening assay system to identifymolecular variants of nephrotoxic compounds that will be taken up andtransported by hOAT to a lesser degree than is the case with theparental compound.

A further object is to identify alleles or isoforms of hOAT that areassociated with sensitivity to nephrotoxic compounds, particularlynephrotoxic drugs.

These and other objects of the invention will be apparent fromconsideration of this specification as a whole.

SUMMARY OF THE INVENTION

In accordance with the objects, applicants provide isolated nucleic acidencoding hOAT and isolated hOAT polypeptide. hOAT is expressed inrecombinant cells, where it finds use in evaluating compounds fornephrotoxicity or for identifying compounds having the ability toprevent nephrotoxicity. Since hOAT is expressed in human brain, it alsofinds use in characterizing compounds known or suspected to influencebrain function. hOAT also is useful in a method for identifying any hOATalleles and isoforms which are correlated with patient sensitivity tonephrotoxic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1–3 depict a nucleotide sequence for cDNA encoding hOAT (SEQ. IDNo. 1) and its translated sequence (SEQ. ID No. 2). FIG. 1 depicts thefirst 780 nucleotides of the sequence. FIG. 2 depicts nucleotides781-1560 of the sequence. FIG. 3 depicts nubleotides 1561-2123 of thesequence.

DETAILED DESCRIPTION OF THE INVENTION

hOAT polypeptide is defined as a polypeptide sequence that is at leastabout 85% homologous by amino acid sequence (ordinarily at least about90%, and prefereably at least about 95%) with the sequence of FIG. 1(reference sequence hOAT).

An expressed sequence tag (EST) of approximately 200 bp having highhomology to a segment of hOAT is found in the GenBank EST entries underaccession no. R25797. Invention hOAT nucleic acids per se as definedherein exclude any expressed sequence tag (EST) or other nucleic acidsequences found in public databases on the filing date (such databasesbeing expressly incorporated by reference), including the sequence ofaccession no. R25797 as well as the rat ROAT1 and OAT1, and mouse NKT,sequences of the prior art. However, other inventive subject matter suchas isolated protein, methods for screening and the like as set forthabove do not (unless expressly stated to the contrary) exclude the useof the R25797 sequence or its expression product.

“Homology” is defined as the percentage of residues in a candidate aminoacid sequence that is identical with the residues in the referencesequence hOAT after aligning the two sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well know in the art. One computerprogram which may be used or adapted for purposes of determining whethera candidate sequence falls within this definition is “Align 2”, authoredby Genentech, Inc., which was filed with user documentation in theUnited States Copyright Office, Washington, D.C. 20559, on Dec. 10,1991.

“Isolated” hOAT nucleic acid is one that has been separated from itsenvironment as it is found in nature, i.e., from the genome in the caseof DNA or from cellular environment in the instance of RNA.

“Isolated” hOAT polypeptide is one that has been separated from itsnormal cellular environment, and includes hOAT protein that ishomogeneous by SDS-PAGE using silver stain.

In calculating amino acid sequence homology the candidate and referencesequences are aligned in the fashion that produces the maximum number ofaligned residues, with insertions and deletions of residues representedby gaps in the aligned sequences. For example, a 120 residue polypeptidecontaining a 100 residue reference sequence fragment fused at itsN-terminus to a 6 residue polyhistidine affinity tag, but with a singlesubstitution in the hOAT domain, is calculated to be 99% homologous tothe hOAT reference sequence since the sequence of the fragmentcorresponds exactly to the maximally aligned hOAT reference sequenceexcept for a single residue substitution and the 6 residue N-terminalfusion. Thus, if the alignment-maximizing comparison of the candidateand reference sequences reveals an insertion (or deletion) of one ormore amino acid residues, then these residues are ignored for thepurposes of making the homology calculation. Applicant recognizes thatthis convention gives rise to theoretical 100% homology between 2differing sequences, but has chosen to establish his own definition forthe purposes of this filing.

Analysis of homology is based on any one or more of the sequencesimputed from the nucleic acid used to express the hOAT, the sequence ofthe product as first produced in vitro, or the sequence after anypost-translational modification. Thus, if the reference and candidatesequences are identical when expressed, but a glutamine residue is laterdeaminated to glutamic acid, the first candidate is 100% homologous, butthe deaminated sequence is not.

For the purposes herein “hOAT activity” means any one or more of thefunctions performed by hOAT in the human, including in particular thetransport of organic anions.

It is not necessary for a polypeptide to have anion transport activityin order to fall under the definition of hOAT herein. For example, insome embodiments hOAT polypeptides possess at least one immune epitopethat is capable of substantial cross-reaction with an antibody raisedagainst reference sequence hOAT, and thus are useful in immunoassays forhOAT, but may possess mutations that render the polypeptide incapable ofanion transport.

The hOAT polypeptides of this invention comprise substitutions for,deletions of, or insertions of any amino acid residue adjacent to any ofthe reference sequence amino acid residue sites shown in FIG. 1.Substitutional hOATs are those in which at least one amino acid residuein the reference sequence has been removed and a different amino acidinserted in its place at the same position. One or more residues aresubstituted.

Alanine is a common substitution for any residue, and is commonly usedin alanine scanning to identify functional residues, but it is withinthe scope of this invention to substitute other residues into the hOATreference sequence. The introduced residues generally are naturallyoccurring amino acids, commonly G, A, Y, V, L, I, S, T, D, E, Q, C, M,N, F, P, W, K, R or H (using conventional single letter code; EP323,149). Suitable residues also include hydroxyproline,beta-hydroxyaspartic acid, gamma-carboxyglutamic acid, hydroxylysine ornorleucine, to be employed as alternatives to their namesakes.

These substitutions may be conservative, i.e., the substituting residueis structurally or functionally similar to the substituted residue.Other substitutions will be less conservative in that they constitute anexchange between different structural or functional classes of residues.For the purposes herein, these classes are as follows: 1.Electropositive: R, K, H; 2. Electronegative: D, E; 3. Aliphatic: V, L,I, M; 4. Aromatic: F, Y, W; 5. Small: A, S, T, G, P, C; 6. Charged: R,K, D, E, H; 7. Polar: S, T, Q, N, Y, H, W; and 8. Small Hydrophilic: C,S, T. Intergroup substitutions generally will have greater effects onprotein function than conservative (intraclass) substitutions. Thus, itis particularly within the scope of this invention to introduceconservative substitutions into hOAT and, if the results are notsatisfactory, to introduce non-conservative substitutions at the sites.Typically, however, proline, glycine, and cysteine substitutions orinsertions into the sequence are not favored. An example of an expressedvariant is a change at codon 498 from AGC to ATC, resulting inexpression of isoleucine in place of serine. Other variants areintroduced into DNA encoding hOAT without resulting in a change inprotein sequence, e.g. from ATC to ATT at codon 453 or from GGG to GGTat codon 491.

hOAT variants are readily identified by methods apparent to the ordinaryartisan. For example, sites shown by alanine scanning to influenceselected biological activity are subjected to saturation mutagenesis toidentify the optimal modification for the activity in question, e.g.selectivity for transport of a particular anion.

hOATs representing combinations of sequence variants are within thescope of this invention. 2, 3, 4, 5, or more substitutions, deletions orinsertions are introduced into hOAT as defined herein. Typically, adeletion of a single residue will be accompanied by an insertion within1 to about 3 residues of the deletion site. Generally, deletions oflarger domains unnecessary for anion transport activity need not beaccompanied by an insertion. The results of individual amino acidsubstitutions are generally additive except when the residues interactwith each other directly or indirectly. They are readily screened usingthe same procedures described in Sweet et al. or Sekine et al. (supra)in order to identify those having the properties of reference sequencehOAT or the desired modified properties.

Included within the scope of this invention are hOATs having one or moreamino acids inserted immediately adjacent to a hOAT amino acid at anyposition in the reference sequence. Insertional hOATs generally willhave a polypeptide structure comprising the sequenceNH₂-PP-A-(X)_(n1)-B-PP-COOH, wherein X is the inserted residue(s) (whichmay be the same or different), n1 is an integer (generally 1–30,typically 1 or 2), either A or B are the designated residue sites forinsertion and PP represents the remainder of the hOAT or a bond at thehOAT N or C terminus.

The invention includes fusions of hOAT and selected antibody recognitionsequences (heterologous polypeptides) for immunoaffinity purification ofhOAT from cell culture, fusions of hOAT sequences with affinity tagssuch as FLAG or polyhistidine, and chimeric sequences (particularlyfusions of hOAT sequence fragments with fragments of other receptors ofthe 12-transmembrane spanning region class).

Also included within the scope of this invention are hOATs in which aglycosylation site is introduced or removed from the reference sequence,whether by substitution, insertion or deletion of one or more amino acidresidues. Such changes will result in the installation or removal of thesequence NXS or NXT, where X can be any residue. Thus, asparagine can besubstituted for any residue located 2 residues N-terminal to serine orthreonine to introduce a glycosylation site. Alternatively, singleglycosylation can be omitted by substituting glycosylated asp with anyresidue, deleting site-adjacent serine or threonine substituting anyresidue into the glycosylation site to perturb the NXS or NST sequence.

Also included within the scope of this invention are deletional hOATs,i.e., hOATs in which one or more amino acid residues of the referencesequence have been removed at a designated site, whereby flankingresidues are now joined by a peptide bond in the ordinary fashion. Itgenerally is not preferred to delete P, C or G residues.

Typically, deletions or insertions are relatively small, on the order of1 to 10 residues and generally no more than 2, although deletions orinsertions can be quite large if they are not in critical portions ofthe reference sequence, or the additional sequence is to be removed atsome point during post-translational or post-recovery processing. Thenumber of residues that are deleted or inserted in part will depend uponwhether or not they are found in secondary structural components such ashelices or sheets (whereupon only 1 or, preferably 2 residues areinserted or deleted), or are in less structurally confined domains suchas loops, where larger numbers of residues may be deleted or insertedwithout unduly perturbing the structure of hOAT.

The hOATs of this invention may be subject to post-translationalcovalent modification, e.g. deamidation of asparagine or glutamine, oroxidation of cysteine residues. Glycosylation can be variant or absentdepending upon the host cell used to express the variant. hOATscontaining such modifications are included within the scope of thisinvention. If hOAT is glycosylated in recombinant cell culture, itpreferably is glycosylated with carbohydrates characteristic ofmammalian cells, although it also may bear fungal (such as yeast)glycosylation patterns. Glycosylation is acceptable which ischaracteristic of expression of hOAT from one or more of fibroblast,kidney, brain, lung, skin, neural, liver or bone marrow cells or celllines, or from any mammalian cell line such as CHO or embryonic kidneycells.

Naturally occurring human alleles are included within the scope of thisinvention. These readily are identified by obtaining nucleic acidsamples from individuals in a population, sequencing hOAT from suchindividuals and determining residues at which variation is found. Onceeach variation is identified, it is straight-forward to determine thefrequency of the putative allele in other individuals by PCR usingprimers specific for the domain in question, or such other methods asare conventional in the field for determining proportions of alleles inhuman populations.

Preparation of hOAT Polypeptides

The hOATs of this invention are readily prepared by methods known in theart. In general, nucleic acid encoding the hOAT is prepared by PCR, byin vitro synthesis (Vasser et al., “N.A.R.” 18(a0):3089 [1990]), bycloning from a human genomic or kidney cDNA library or combinationsthereof. Site-directed mutagenesis of hOAT-encoding nucleic acid is usedto prepare nucleic acid encoding sequence variants. The + (coding) and −strands (antisense) of hOAT are included within the scope of hOATnucleic acids, as are hOAT-encoding cDNA, genomic DNA and RNA. hOAT DNAincludes 5′ and 3′ regions of the hOAT gene that are not transcribed butserve as transcription control domains, and transcribed but notexpressed domains such as introns (including splice junctions),polyadenylation signals, ribosomal recognition domains and the like.

The hOAT nucleic acid is expressed in in vitro systems or in recombinanthost cells. One method for expression is ribosome based synthesis usingdedicated tRNAs (Benner, “TIBTECH” 12:158–163 [1994] and Robertson etal., “J. Am. Chem. Soc.” 113:2722–2729 [1991]). Ordinarily, however, thehOAT-encoding nucleic acid (generally DNA) is inserted into anappropriate expression vector recognized (transcribed and translated) bythe host cells, host cells are transfected with the expression vector,the recombinant host cells are grown in suitable culture medium, andoptionally the desired hOAT is recovered from the recombinant cellculture by chromatographic or other purification methods. It is alsowithin the scope of this invention to partially synthesize the hOAT inrecombinant cell culture or by in vitro methods and then ligate thepolypeptide fragments by peptide ligase (reverse proteolytic synthesis).

The nucleic acid for expression of the hOAT may comprise an exogenoussignal sequence. Here, the hOAT is expressed as a preprotein of hOAT,whereby the hOAT is expressed as a precursor that is processed to maturehOAT. Suitable presequences include those of (a) microbial proteasessuch as subtilisin, (b) mammalian polypeptides, (c) cytokines such asgamma interferon or an interleukin, (d) growth factors such as growthhormone or TGF-alpha, (e) polypeptides or proteins having N-terminalmature sequences that are homologous to human hOAT, (f) immunoglobulins,(g) receptors, or (h) other presequences of secreted or cell membranebound proteins. Signal sequences optionally are derived from or arehomologous to the host cell, or at least the phylogenetic branch towhich the host cell belongs. For example, one ordinarily would use apresequence of a yeast protein, such as mating factor, in a yeastexpression system, or of a bacterium, such as ST-II or beta-lactamase,in bacterial cell culture systems. A wide variety of suitable signalsequences are known and can be used in methods for the preparation ofthe hOATs described herein.

The nucleic acid constructs encoding hOAT generally are spliced intoexpression vectors where they will be under the control of sequencescontrolling transcription, translation and RNA stability. Thesesequences include promoters, operators, enhancers and polyadenylationsequences, and are generally known in the art. Constructing suitableexpression vectors for hOATs of this invention is a matter of routinefor those skilled in the art, and will be accomplished using theconventional tools of molecular biology, including nucleic acidsynthesis in vitro, PCR, adapters, ligases, restriction enzymes,expression and shuttle plasmids, transfection aids and the like, all ofwhich are publicly (and for the most part commercially) available.

Suitable host cells for transfection with nucleic acid encoding hOAT arewell known. Some are mentioned above while others are described in WO93/13208 at page 12, line 21–page 19, line 5, and EP 319,312 B1, page16, lines 10–18 and Table II thereof. It may be optimal to use hostcells that are capable of glycosylating hOAT, typically includingmammalian cells such as embryonic kidney 293 cells, COS cells, CHO,BHK-21 cells and the like. Xenopus oocytes are suitable for expressionof hOAT RNA. In addition, host cells that have been used heretofore toexpress anion transporter polypeptides in recombinant cell culture aresuitable.

The host-vector system should yield substantially homogeneous hOAT,thereby avoiding the need to purify various hOAT alleles, isoforms orcleavage products from one another. If the host cell is capable ofglycosylation, essentially all of the hOAT molecules should beglycosylated. In addition, host cells optimally will not proteolysehOAT. Cells can be selected that contain no protease, e.g., in theperiplasm, that will cleave hOAT. For example, E. coli and othermicrobial strains are known that possess little or no extracellular orperiplasmic proteolytic activity (other than signal peptidases). Theabsence of deleterious proteases helps to ensure that the product is notrendered microheterogenous as to chain length by host-endogenousproteases acting on the hOAT expression product. In addition, oralternatively, basic residues of hOAT that define sites for proteolyticcleavage are substituted with residues other than K or R.

The hOAT recombinant cells are cultured under conventional conditionsusing conventional culture media heretofore employed with the cells inquestion. These conditions and media are widely known. Freshlytransfected cells may only transiently express the hOATs, but stabletransformants readily are obtained in accord with conventional practiceusing cotransformation with a selection gene such as DHFR or glutaminesynthetase and serial culture in the presence of a selection agent suchas methotrexate or methionine sulfoximine, respectively. Yields of hOATscan differ substantially despite minor differences in the character ofsubstituents or insertions. In such cases, it is desirable to screen foran expression system that will yield a quantity of hOAT that is at leastabout 75% of that obtained with the reference hOAT in the sameexpression system.

The hOAT may be expressed in bacteria in the form of retractile bodies,in which base the insoluble hOAT is recovered and refolded using knownmethods, e.g. dissolution in a denaturant such as guanidiniumhydrochloride followed by gradual removal of the denaturant. Directlyexpressed hOATs of this invention may have an extra N-terminalmethionine or blocked methionine residue, although host cells can beemployed that will cleave away such N-terminal methionine residues ifthey are extraneous to the protein as found in nature. In order to avoiddifficulties with insoluble expression products it is preferable toexpress hOAT in eukaryotic, more preferably mammalian, cells.

hOAT is isolated or purified from recombinant cell culture by methodsheretofore employed for other proteins, e.g., native or reducing/SDS gelelectrophoresis, isoelectric focusing, immobilized pH gradientelectrophoresis, salt precipitation, solvent fractionation (usingethanol for example) and chromatography such as gel filtration, ionexchange (cation or anion), ligand affinity (cibacron blue F3GA orp-aminobenzamidine), immunoaffinity, chromatofocusing, reverse phase orhydrophobic interaction chromatography. Typically, the hOAT will beisolated so as to be >95% pure by weight of protein, and preferablygreater than 99% pure. However, the term “isolated” as used in referenceto hOAT protein or nucleic acid refers to the absence of one or more ofthe normal human polypeptides or nucleic acids found in association withhOAT in its natural environment, and does not necessarily imply that thehOAT is purified to any degree free of non-hOAT proteins. Since hOAT isnormally found in the cell membrane, it is preferable to recoverrecombinant hOAT as a membrane preparation; otherwise the function ofhOAT may be perturbed. In any case, many utilities for hOAT do notrequire purification of the protein at all; screening assays foragonists or antagonists are best conducted in intact host cells.

The hOAT polypeptides or their fragments optionally are prepared invitro, especially if they are relatively small, e.g. on the order ofabout 30 residues or less. For example, hOATs are prepared by synthesisusing standard solid-phase peptide synthesis procedures as described byMerrifield “J. Am. Chem. Soc.” 85:2149 (1963). These then are ligatedtogether by the use of peptide ligase reverse proteolysis). In vitromethods of protein synthesis also are used to prepare hOATs without theneed for recombinant cell culture. Such methods are useful forsmall-scale preparations, and have the advantage of reducing thepossible effect on yields of host cell proteases. In vitro hOAT proteinsynthesis has one additional quite substantial advantage in that itpermits the site-specific introduction into the hOAT of a non-naturallyoccurring amino acid residue. Accordingly, when the term “amino acidresidue” is used herein (in connection with hOAT modification bysubstitution or insertion, especially by a single amino acid) it will beunderstood that the amino acid is not limited to the naturally occurringresidues associated with native tRNAs. Aminoacyl tRNA is efficientlyprepared using a variety of non-naturally occurring amino acid residues(“non-naturally occurring” means not naturally found in proteins,although the amino acid might be found elsewhere in nature). Since thetRNA is selected to be incorporated at a codon not recognized by any ofthe tRNAs normally involved in protein synthesis, the selectednon-naturally occurring amino acid residue is incorporated only at theparticular site in the hOAT sequence chosen for the unique codon. Thus,in these cases the hOAT is encoded by a nucleic acid having a nonsensecodon, e.g., UAG, at the desired unique insertion or substitution site.

Suitable non-naturally occurring amino acids are described for examplein Greenstein et al., “Chemistry of the Amino Acids” Vols. 1–3, 1986. Ingeneral, one will use pharmaceutically innocuous L-amino acids that arefound in nature but ordinarily not incorporated into proteins. Suchamino acids typically will be structurally related to a naturallyoccurring residue that produces the desired effect at a given site andwill be used to further resolve and optimize the desired property of thehOAT.

The hOATs of this invention also include hOATs that have beensubstituted by a non-peptidyl moiety, either for purposes of preparingthe hOAT to begin with or as a subsequent modification of the hOATprepared by amino acid substitution, insertion or deletion as describedelsewhere herein. Covalent modification may accomplish essentially thesame objective as a site-directed mutant at the same location using anaturally occurring residue.

Tryptophan is a relatively rare amino acid in hOAT. Accordingly, thisresidue is an attractive site for post-translational convalentmodification because substitution at other sites is expected to be lessthan may be the case with more common residues. Reaction of trp with anoxidant such as a halogen donor, e.g. bromine, will yield the side chainstructure

This reaction should be conducted in aqueous solvent and at low halogenconcentrations.

Other covalent modifications of hOATs will be apparent to the artisan.Sensitive side chains are protected by masking them with antibodiesdirected against an epitope that includes the residue to be protected.Reagents for accomplishing such modifications are well-known and havebeen widely used in the diagnostic and preparative fields. See T.Creighton, Proteins: Structure and Molecular Properties, 1983.

hOATs are cross-linked to a water insoluble matrix or incorporated intoa lipid vehicle such as a liposome, usually a ULV. Cross-linking isaccomplished by reacting the hOAT and matrix with a bifunctional agent.Examples of suitable bifunctional agents include1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dethiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, hOAT is immobilized on reactive water-insolublematrices such as cyanogen bromide-activated carbohydrates and thereactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; and 4,330,440.

hOATs also are covalently modified by linking the hOAT to variousnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol or polyoxyalkylenes, in the manner set forth forexample in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337. PEG is a non-immunogenic, linear, unchargedpolymer with three water molecules per ethylene oxide unit. SeeMaxfield, et al., “Polymer” 16:505–509 (1975); Bailey, F. E. et al., in“Nonionic Surfactants”, Schick, M. J., ed, pp. 794–821 (1967). Severaltherapeutic enzymes have been conjugated to PEG to increase their invivo half-life (Abuchowski, A., et al., “J. Biol. Chem.” 252:3582–3586(1977); Abuchowski, A. et al., “Cancer Biochem. Biophys.” 7:175–186(1984). An IL-2 (interleukin-2)-PEG conjugate has been reported toincrease circulatory life and potency (Katre, N. V. et al., “Proc. Natl.Acad. Sci.” 84:1487–1491 (1987); Goodson, R. et al., “Bio/Technology”8:343–346 (1990)). See also Abuchowski, A. et al., “J. Biol. Chem.”252:3578–3581 (1977). Any of the methods for PEG conjugation used inthese citations is acceptable for use with the hOATs of this invention.

Uses for the hOATs of this Invention

The hOATs of this invention are useful in therapeutic, diagnostic andpreparatory methods. Their use will depend upon the properties that theypossess, as will be apparent to the ordinary artisan. For the most part,all of the hOATs will retain hOAT immune epitopes, so they are useful inplace of hOAT in hOAT immunoassays whether or not they possess any aniontransport activity.

The hOATs of this invention are useful in identifying substances thatbind to hOAT (hOAT binding partners, or “TBP”). Of particular interestare substances that are capable of binding to hOAT to substantiallyinhibit the anion transport activity of hOAT, or, preferably, tointroduce favorable selectivity into the transport activity such thatnephrotoxic drugs such as amphotericin or cidofovir are not taken up asavidly as in the absence of the substance. TBP's ability to bind to hOATalso is useful in methods for recovering hOAT from contaminated mixturessuch as cell culture supernatants of recombinant hOAT-expressing cells(including the hOAT amino acid sequence hOATs herein). Typically, hOATscan be used in place of hOAT standards in immunoassays for hOAT if theypossess at least one hOAT epitope recognized by the antibody used in thehOAT immunoassay in question, while the TBPs are used in place ofantibodies for hOAT. The hOATs also are useful, as appropriate, infunctional assays for certain individual properties of hOAT.

Peptide TBPs are obtained by the use of in vitro directed evolutionarymethods such as those employing filamentous phage to present candidatesequences (otherwise known as phage display) and similar methods knownper se which rely on the systematic generation and screening of peptidesfor activity. These typically are rather small molecules, containing onthe order of about 5 to 20 residues.

Antibody TBPs are immunoglobulins, ordinarily monoclonal antibodies,which (in preferred embodiments) are capable of specifically inhibitingthe transport function of hOAT. Antibodies are raised in conventionalfashion by immunizing an animal with an immunogenic hOAT conjugate, e.g.hOAT crosslinked to keyhole limpet hemocyanin.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are essentiallyidentical in specificity and affinity. Monoclonal antibodies includehybrid and recombinant antibodies (e.g. “humanized” antibodies)regardless of species of origin or immunoglobulin class or subclassdesignation, as well as antibody fragments (e.g., Fab, F(ab′)_(2′), andFv). Thus, “monoclonal” antibodies are produced by any particular methodthat will yield a substantially homogeneous population. For example,monoclonal antibodies may be made using the methods described by Kohler& Milstein, “Nature” 256:495 (1975), Goding, Monoclonal Antibodies:Principles and Practice pp. 59–103 (1986), Kozbor, “J. Immunol.”133:3001 (1984), or Brodeur, et al., Monoclonal Antibody ProductionTechniques and Applications, pp. 51–63 (1987), or may be made byrecombinant DNA methods. Cabilly, et al., U.S. Pat. No. 4,816,567.

In a preferred embodiment of the invention, the monoclonal antibody willhave an affinity for reference sequence hOAT of at least about 10⁹moles/liter, as determined, for example, by the Scatchard analysis ofMunson & Pollard, “Anal. Biochem.” 107:220 (1980). Also, the monoclonalantibody typically will inhibit the transport activity of hOAT (using astandard organic anion such as para-aminohippurate) by at least about50%, preferably greater than 80%, and most preferably greater than 90%,as determined by conventional methods.

DNA encoding the monoclonal antibody is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). Hybridoma cells serve as a preferredsource of such DNA. Once isolated, the DNA may be placed into expressionvectors, which are then transfected into host cells such as simian COScells, Chinese Hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells.

The DNA optionally may be modified in order to change the character ofthe immunoglobulin produced by its expression. For example, humanizedforms of murine antibodies are produced by substituting acomplementarity determining region (CDR) of the murine antibody variabledomain for the corresponding region of a human antibody. In someembodiments, selected framework region (FR) amino acid residues of themurine antibody also are substituted for the corresponding amino acidresidues in the human antibody. Humanized forms of murine antibodiesalso may be produced by substituting the coding sequence for human heavyand light constant chain domains in place of the homologous murinesequences. Morrison, et al., “PNAS” 81:6851 (1984).

TBP also includes nucleic acid sequences that bind hOAT. Evolutionaryselection methods for oligonucleotides that bind to target proteins arewell known (WO 92/14843; Ellington et al., “Nature” 355:850 (1992); Bocket al., “Nature” 355:564 (1992); Ellington et al., “Nature” 346:818(1990); Tuerk et al., “Science” 249:505 (1990). These oligonucleotides,commonly known as aptamers, generally contain the usual A, T, G, C or Ubases or derivatives thereof, and comprise sequences that bind to apredetermined site on a target protein. A selection method for TBPs thatinhibit the transport function of hOAT comprises (a) preparing a pool ofcandidates (oligonucleotides, peptides, extracts, proteins, etc.), (b)contacting the candidates with hOAT having anion transport activity(typically reference sequence hOAT) (c) isolating from the hOAT thosecandidates that are able to bind to hOAT, (d) contacting the candidatesfrom step c) with an hOAT in which the hOAT transport function has beenmutated substantially out of the hOAT, and (e) recovering thosecandidates that do not bind to the mutated form.

For diagnostic applications, the hOATs of this invention optionally arelabeled with a detectable moiety, either in vivo or in vitro. Thedetectable moiety can be any substituent which is capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; a radioactive isotopic label,such as, ¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art per se can be used to conjugate the hOAT tothe detectable moiety. See the methods described supra and Hunter, etal., “Nature” 144:945 (1962); David, et al., “Biochemistry” 13:1014(1974); Pain, et al., “J. Immunol. Meth.” 40:219 (1981); and Nygren, J.“Histochem. and Cytochem.” 30:407 (1982). Oligonucleotide TBPs arelabeled in the conventional fashion heretofore employed in thediagnostic probe art.

The TBPs or hOATs of the present invention optionally are employed inknown immunoassay techniques, such as competitive binding assays, directand indirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147–158 (1987).

Competitive binding assays rely on the ability of a labeled standard(which may be hOAT, or an immunologically reactive portion thereof suchas a labeled hOAT of this invention) to compete with the test samplehOAT for binding with a limited amount of TBP. The amount of hOAT in thetest sample is inversely proportional to the amount of standard thatbecomes bound to the TBP. To facilitate determining the amount ofstandard that becomes bound, the TBP generally is insolubilized beforeor after the competition, so that the standard and analyte that arebound to the TBP conveniently are separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two TBPs, each capable of binding toa different target portion, or epitope, of hOAT. In a sandwich assay,the test sample analyte is bound by a first TBP which is immobilized ona solid support, and thereafter a second TBP binds to the analyte, thusforming an insoluble three part complex. David & Greene, U.S. Pat. No.4,376,110. The second antibody may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using an anti-TBPantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

The TBPs of this invention also are useful for in vivo imaging, whereina TBP labeled with a detectable moiety is administered to a host,preferably into the bloodstream, and the presence and location of thelabeled TBP in the host is assayed.

hOAT nucleic acids or TBPs also can be used in a method for identifyingisoforms or alleles of hOAT nucleic acids or polypeptides that place apatient at particular risk for nephrotoxicity. Such variants, alleles orisoforms will exhibit different selectivity towards uptake of certainanions. If these anions are nephrotoxic drugs or drug metabolites, thena patient may be at particular risk of injury. The patient can be tested(e.g., by amplifying genomic hOAT-encoding DNA from a blood sample) todetermine if the at-risk variant, isoform or allele is present. If so,then the dosage of the drug can be reduced or an alternative drug used.This method comprises identifying one or more naturally-occurringsequence variations in the human population and determining theselectivity of such variations for transport of a selected nephrotoxicsubstance. Then a patient is tested for the variation associated withrelatively selective transport of the nephrotoxic substance.

Similarly, allelic variation in the transcriptional control regions ofthe hOAT gene can be analyzed for correlation with susceptibility touptake and transport of selected nephrotoxic drugs; here the correlationwill be with the amount of expression of hOAT as opposed to theselectivity of the hOAT protein. This method comprises identifying oneor more naturally-occurring sequence variations in the expressioncontrol domain of hOAT in the human population and determining theexpression levels of hOAT in cells containing such variant. Patientshaving relatively high levels of expression of hOAT may not beconsidered suitable for treatment with nephrotoxic drugs, or the dosageof such drugs may be reduced.

Since at least a domain of hOAT is expressed in the brain as well as thekidney (cf brain EST R25797 and Example 3 below), it is also within thescope of this invention to probe a brain library to obtain the fulllength gene corresponding to R25797. The sequence of this gene may ormay not diverge substantially from the reference sequence hOAT. Thisnotwithstanding, the brain-expressed gene is also a suitable subject fordetermination of sequence variants such as alleles and isoforms. Thesemay be involved in important neural functions and are suitable subjectsfor screening to identify them.

hOAT also is useful in screening methods for compounds that can act tosuppress or enhance anion uptake and transport by hOAT. Suppression ofhOAT activity (antagonism) is useful in reducing the nephrotoxicity ofdrugs, i.e., to serve as nephroprotective agents, while enhancement ofhOAT activity (agonism) is useful in the treatment of kidneydysfunction. The screening methods comprise providing a candidate hOATagonist or antagonist, contacting the candidate with hOAT nucleic acidor polypeptide, determining the effect of the candidate on thetranscription of the hOAT nucleic acid (or the expression or biologicalactivity of the hOAT polypeptide), identifying a hOAT agonist orantagonist, and optionally preparing additional quantities of theagonist or antagonist that is so identified. Biological activity of hOATis assayed using the stable cell line of Example 1 as shown in Example2.

hOAT is useful in screening assays for modified forms of heretoforenephrotoxic drugs, or as part of a toxicology screening program todetermine the potential nephrotoxicity of new therapeutic compounds.Accumulation of the candidate in hOAT transformants as opposed tocontrol cells is an indicia of potential nephrotoxicity. DeterminingCC₅₀'s using the hOAT transformants would produce a direct measure ofpotential nephrotoxicity. A candidate form of a suspected compound or aknown nephrotoxic drug, e.g., a prodrug, is contacted with the hOAT andits effect on the transport by hOAT of a benchmark anion is determined.Transport of the test compound itself (usually in an isotopicallylabeled form) also can be determined. Alternatively, transport of thecompound in the presence of probenecid can be assayed. If the compoundis no longer transported, or the benchmark anion transport is notaffected by the compound, or probenecid has no influence on whether ornot the compound is transported, then the candidate is suitable forfurther study in animals as a potential non-nephrotoxic drug, forexample, in the case of a prodrug as a non-nephrotoxic form of theparental drug. Prodrugs of nucleotide phosphonate analogues such ascidofovir, PMEA or PMPA suitable for testing include mono and di-estersand amidates of the phosphonyl group.

EXAMPLE 1 Generation of a Cell Line Stably Transformed with hOAT1 Gene

A cell line stably expressing hOAT1 was prepared and characterized toultimately prove that the isolated cDNA encodes functional hOAT1protein.

The expression construct containing hOAT1 gene was prepared as follows.hOAT1 coding sequence was PCR amplified from plasmid isolated from humankidney cDNA library. PCR reaction was performed under standardconditions using Expand High Fidelity PCR system (Boehringer Mannheim).Oligonucleotides5′-ACCGTCTAGAATTCTTTTTATTTTTAATTTTCTTTCAAATACGTCCACCATGGCCTTTAATGACCTCCTGCAGCAGG-3′(SEQ. ID NO. 3) and5′-TACTCACGTGGATCCTGATCAGACGTCTGTAGGACCTTCCCTCCCTTTAGG-3′ (SEQ. ID NO.4) were used as a sense and antisense primer to introduce EcoRI andBamHI restriction site, respectively. In addition, the sense primercontained truncated 5′-untranslated sequence of alfalfa mosaic virus(underlined) and favorable Kozak consensus sequence (CCACCATGG) (SEQ. IDNO.5) to maximize initiation of translation. The PCR product wasdigested with EcoRI/BamHI and cloned into pIRESneo expression vector(CLONTECH, Palo Alto, Calif.) using the corresponding restriction sitesto yield pIRES-hOAT plasmid. After cloning, the correct nucleotidesequence of the whole fragment generated by PCR was verified.

pIRES-hOAT plasmid was transfected into CHO-K1 cells (ATCC CCL61)growing in F-12 nutrient mixture supplemented with 10% fetal bovineserum, 100 U/ml penicillin, and 100 μg/ml streptomycin. 24 hours beforetransfection, approximately 6×10⁶ CHO-K1 cells were seeded into 100-mmPetri dish. At the time of transfection, media was aspirated and 6 ml offresh media containing 12 μg of pIRES-hOAT and 60 μg of Cytofectin GSVcationic lipid (Glen Research, Sterling, Va.) was added to the cells.Following overnight incubation, selection of stable transformantsstarted in phenol red-free F-12 nutrient mixture containing fetal bovineserum, penicillin and streptomycin as above, plus 1 mg/ml G418(CLONTECH). Viable colonies were isolated after 2-week selection andtested for uptake of p-aminohippuric acid (PAH, a prototype substrate oforganic anion transport systems) in the presence and absence ofprobenecid (inhibitor of organic anion transport). For the PAH uptakeassay, the cells were seeded into 12-well plates at the density of 2×10⁵cell/well. After 48 hours, growth media was aspirated and the cells werewashed twice with phosphate buffered saline (PBS). The uptake assay wasperformed in transport buffer (135 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2mM MgCl₂, 0.8 mM MgSO₄, 28 mM glucose, and 13 mM HEPES pH 7.2)containing 5 μM [³H]PAH (New England Nuclear, spec. activity 1.2Ci/mmol) ±1 mM probenecid. After 90 min incubation at 37° C. in thetransport buffer, the cells were washed 3-times with ice-cold PBS (2ml/well) and lysed directly on the plate in the presence of 0.3% TritonX-100 (0.5 ml/well) for 20 min at room temperature. Plates were washedwith additional 0.5 ml/well of Triton X-100, the lysate and wash werecombined, scintillation fluid was added, and the radioactivity counted.As a result of this assay, a transformed clone with 30-fold increase inPAH uptake compared to parental CHO-K1 cells was identified anddesignated CHO-hOAT.

EXAMPLE 2 Functional Characterization of hOAT1

Kinetics of hOAT1-mediated PAH uptake was characterized using CHO-hOATcells. The uptake assay was performed as described above with variousconcentrations of [³H]PAH ranging from 5 to 160 μM. To assess nethOAT1-specific uptake, background uptake measured in parental CHO-K1cells at each substrate concentration was subtracted from thatdetermined in CHO-hOAT cells. Kinetic constants were calculated usingEnzyme kinetics software (ChemSW, Fairfield, Calif.). As expected, PAHuptake in CHO-hOAT cells was saturable with K_(m)=13 μM and Vmax=42pmol/10⁶ cells. As oppose to the background uptake of PAH in CHO-K1cells, uptake in CHO-hOAT cells was strongly sensitive to probenecidwith IC₅₀=6.2 μM when measured at PAH concentration equal to its K_(m).In addition, PAH accumulation in CHO-hOAT was stimulated approximately2.5-fold by preloading the cells with 10 mM glutarate indicating thatthe hOAT protein functions as an organic anion/dicarboxylate exchanger.

EXAMPLE 3 Tissue Distribution of hOAT1

A human multiple tissue Northern blot (CLONTECH) was used forlocalization of hOAT1 expression in human tissues. A hOAT1-specific[³²P]dATP labeled probe was generated by random priming the BsrGI/Bsu36IDNA fragment corresponding to nucleotides 420–854 of the hOAT1 codingsequence. The membrane was hybridized for 1 hour at 68° C. in ExpressHybhybridization buffer (CLONTECH) and then washed twice in 2×SSC with0.05% SDS for 30 minutes at room temperature followed by a single washin 0.1×SSC with 0.1% SDS at 50° C. A strong signal was detected inkidney corresponding to a 2.5 kb hOAT1 transcript. No positive signalwas found in the other tested tissues (brain, heart, skeletal muscle,colon, thymus, spleen, small intestine, placenta, lung, or peripheralblood leukocytes).

In addition, hOAT1 expression in various human tissues was examined bymore sensitive RT-PCR amplification. For this purpose, the MultipleChoice cDNA kits I and II containing tissue specific cDNAs (OrigeneTechnologies, Rockville, Md.) and two sets of hOAT1-specific primerswere used for PCR detection of hOAT1 expression. The oligonucleotides,

-   -   5′-CCCGCTGGCACTCCTCCTCCGGGAG-3′ (SEQ. ID NO. 6) (sense), and    -   5′-GTAGAGCTCGGCAGTCATGCTCACCA-3′ (SEQ. ID NO. 7) (antisense),        was used to amplify a 606-bp fragment from the hOAT1 coding        region (nucleotides 815–1420). In the independent set of PCR        reactions, a 295-bp hOAT1 fragment (comprised of the last 175        coding nucleotides and 120 nucleotides of the 3′-UTR) was        amplified using the oligonucleotides,    -   5′-CCAGCGCTGTCACTGTCCTCCTGC-3′ (SEQ. ID NO. 8) (sense), and    -   5′-AACCCCCACACTTGGGTCACCATTTCCTC-3′ (SEQ. ID NO. 9) (antisense).        PCR reactions were carried out using the Expand High Fidelity        PCR System (Boehringer Mannheim) in a total volume of 25 μl        containing 1 μg of tissue-specific cDNA. Thirty-five        amplification cycles (95° C. for 40 s, 58° C. for 1 min, and        72° C. for 45 s) were performed. Positive tissues were        identified after separation of PCR reactions on a 1% agarose        gel. As expected, a strong positive signal was consistently        detected in kidney. In contrast with the Northern analysis,        brain and skeletal muscle was also positive for hOAT1 expression        although to lesser extent compared to kidney.

Presence of hOAT protein in human kidney tissue was verified by animmunoblot analysis with rabbit anti-hOAT1 polyclonal antibody preparedat AnaSpec (San Jose, Calif.) using standard immunological techniques.Briefly, an immunogenic peptide, NH2-TVQDLESRKGKQTR-COOH (SEQ. ID NO.10), corresponding to hOAT1 amino acids 515–528 was conjugated tokeyhole limpet hemocyanine through a cysteine residue added to thepeptide's C-terminus. Animal serum was collected after 4 immunizationsin the presence of complete Freund's adjuvant and affinity-purifiedagainst the immunogenic peptide immobilized on a Sepharose resin. Forimmunoblot analysis, human kidney cortex was extractracted with a buffercontaining 20 mM Tri-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5%deoxycholate, and 0.1% SDS. After homogenization in the presence ofcomplete proteinase inhibitor cocktail (Boehringer Mannheim) the extractwas clarified by high-speed centrifugation, separated by electrophoresison an 8% SDS-polyacrylamide gel and electroblotted onto a nitrocellulosemembrane (Millipore, Bedford, Mass.). The membrane was blocked in PBS/5%dry milk (PBS-M) for 1 hour, washed 3 times in PBS/0.05% Tween 20(PBS-T), and incubated overnight in PBS-M with the anti-hOAT antibody.Following wash in PBS-T, the membrane was incubated in PBS-M with goatanti-rabbit antibody conjugated to horseradish peroxidase (Zymed, SouthSan Francisco, Calif.). After an additional wash and incubation with achemiluminescent substrate (Amersham, Arligton Heights, Ill.), theimmunoblot was exposed to X-ray film. The antibody recognized aheterogeneous product with an apparent molecular weight of 80 to 90 kDa.This was significantly larger than the predicted molecular weight fromthe hOAT1 amino acid sequence (60.3 kDa). However, when the cortexextract was treated with peptide:N-glycosidase F, which specificallycleaves N-linked oligosaccharide chains, a homogeneous product of 60 kDawas detected on the immunoblot.

EXAMPLE 4 Determination of Nephrotoxic Potential by Cell Culture AssayUsing hOAT Transformants

Adefovir (ADV) is an anti-HIV nucleotide analog with unique resistanceprofile currently undergoing Phase III clinical evaluation. The mostimportant clinical toxicity of ADV is nephrotoxicity associated withchanges in laboratory markers of renal functions. ADV is a substrate forhuman renal organic anion transporter 1 (hOAT) located in thebasolateral membrane of the proximal convoluted tubules. In thisexample, the role of hOAT in the mechanism of ADV nephrotoxicity wasinvestigated.

Chinese hamster ovary cells (CHO), which exhibit low sensitivity to ADVcytotoxicity due to its limited uptake, were stably transformed withhOAT cDNA to generate CHO-hOAT cells as described above. Uptake andcytotoxicity of ADV in the two cell lines was compared.

CHO-hOAT accumulated ADV to levels >300-fold higher compared to CHO.Uptake of ADV by CHO-hOAT was saturable (K_(m)=23 uM, V_(max)=390pmol/10⁶ cells) and sensitive to the hOAT inhibitor probenecid (PBC;IC₅₀=6.5 uM). Importantly, ADV was ˜500-fold more cytotoxic in CHO-hOATcompared to CHO cells. However, in the presence of 1 mM PBC, CHO-hOATwere only 3-fold more susceptible to ADV than were CHO. Anotherantiviral nucleotide, cidofovir (CDV), but not its cyclic prodrug(cCDV), also efficiently accumulated in CHO-hOAT (K_(m)=70 uM,V_(max)=1,110 pmol/10⁶ cells). Accordingly, CDV was >400-fold morecytotoxic in CHO-hOAT compared to CHO. In contrast, cytotoxicity of cCDVwas increased to much lesser extent in CHO-hOAT cells corresponding withthe lack of cCDV nephrotoxicity. Similar to ADV, CDV cytotoxicity wasalso significantly reduced by PBC.

Expression of hOAT enhances cytotoxicity of ADV. Since high-levelexpression of hOAT is specific to renal tubules, hOAT plays a crucialrole in the mechanism of ADV nephrotoxicity. Thus, hOAT inhibitors (e.g.PBC) may be useful to overcome ADV nephrotoxicity. Observations with CDVand cCDV correlate with their nephrotoxic potential and provideadditional support for the involvement of hOAT in this process.

1. An isolated hOAT polypeptide having the sequence of SEQ. ID. NO. 2.